We have recently shown that DNA polymerase beta (Pol ss) plays a key role in maintaining genomic stability and preventing cellular transformation. Approximately 30% of human tumors studied express Pol ss variants. We have shown that several of these tumor-associated variants of Pol ss induce cellular transformation in immortalized cells by a mutational mechanism. Pol ss functions in base excision repair (BER) to fill in gaps that are created during the excision of DNA damage that arises at the rate of 20,000 lesions per cell per day. Thus, accurate DNA synthesis by Pol ss is critical for the maintenance of genomic stability and the prevention of human cancer. The broad, long-term objective of the proposed research is to characterize the fidelity mechanisms of DNA polymerase beta at a molecular level. The specific aims are to test the hypothesis that amino acid residues of Pol ss that are critical for accurate DNA synthesis are located within regions of Pol ss other than the hydrophobic hinge, loop II, and the region between HhH motifs and to study mechanisms used by specific regions of Pol ss that are critical for accurate DNA synthesis. We will take a combined genetic, biochemical and biophysical approach to address these hypotheses. We will use transient-state kinetics to characterize Pol beta mutator mutants that we have obtained via genetic screens. We will also employ fluorescence assays to characterize how the motion of the Pol beta protein affects fidelity. Finally, crystal structures of various Pol beta mutator mutants will be solved in order to relate our biochemical findings to enzyme structure.